New progestational agents



, 2,816,121 Patented Dec. 10, 1957- 021 OA'C 7 2,816,121 I ----oEcn v aoo 1 A NEW PROGESTATIGNAL AGENTS i David H. Gould, Leonia, Hershel L. Herzog, lMountain 1 View, and Emanuel B. Hershberg, West Orange, N. L, if V Collidine assignors to Schering Corporation, Bloomfield, 3., -a l N corporation of N ew ,J erstey k g f g o A 1 No D i pp atio Ja u ry 5, 1957., v

Serial No. 636,230 7 la sl- 1 0 39745) 0A0 R 1 ---0= JH Larg This invention relates to a new group of steroid com- Ho pounds which possess valuable therapeutic properties, and to meth ds f P ep ri am Mo e Particularly, this F. dehydroinvention e es to a g up of =k6t0-1,A,61 0 w-- sews Firms and the fiuo o al gue w ch possess var. hydro- ,l7e-e hin l-l fl-hydm y gro ps nd ither a keto or lytiws yd oxy gr up a t e i n- 7 VII v11:

Th omp unds of ur in en n m y be represented by mg fongwing formula: In the foregoing sequence, the 3-keto group of 4- androstene-l113-ol 3,17-dione (I) is selectively protected, such as by forming the eneamine (II) permitting ethinaacetylide in anhydrous ether followed by hydrolysis yields l7u-ethinyl-4 androstene-l lp-diol-3-one (III-L, A-ring, dehydrogenation transforming III to IV is conveniently elfected microbiologically by means of B, sphaericus according to the analogous procedure described in co-pending application of Charney et -al., Serial No. 570,210, filed March 8,- 1956. The diacetate (V) is formed in wherein X is a member of the group consisting of hydrothe usual manner, and is brominated with an agent such gen and fluorine, and wherein Y is a member of the as N-bromo-succinimide to forrn 6-bromo 17a-ethiny1- group consisting of 1,4 androstadiene 11 8,17/3 d1o1-3-one-11,17-diacetate OH (VI). ,Dehydrobromination, by refluxing with a tertiary and amine such as 'collidine, yields the 1,4,6-androstatriene 1 11,17-diacetate (VII). The diacetate -'(VII,) may be 40 saponified chemically or, preferably, microbiologically The new andrqstatrienes of the general jorrnulaexhibit v by means of the m croorgamsm Flavobacteriwa dehyprogestat iqnal properties and ar n e f l i th q t drogenans var. hydrolytzcum according to the analogous f postmenopausal synd qms, i f qfilq uterus d h procedure described in co-pendmg application of Charn'ieiistifil d so e s uc a ameno hea. Those cornney, Serial No. 458,661, filed September 27, 1954, yield- 5iiii1's" g 1ai i1i an a fluoro group, exhibit a, higher 4 mg 17a-eth1nyl 1,4,6-androstatr1ene-1118,175-diol-3-one a re o t r pe t a i i y h n h noa hallogenated analogues. All the compounds of the invention are ti e Compound VIII nay also be prepared byemploymg Q15 j' 'gd ijfii q the foregoing react ons in a difierent sequence, Forexkq f this invention may p epa -ed by arnple, the p1'0t6CllVe dlacetate' 9f II]; l7? pa i b of gltelfngtjye rouge l in 1 7 thi b prior to halogenatron and dehalogenan vt fdrrn I '1',4,' 's', an1r 3s1m1e ,11 ,1 1,a-di01-3=one we. prefer to em etmny1-4,6,androstad1en9* 1 o de lav-:1 ;Qlq'f {5g f 11" L i sqquenqg Qf gactiqns; acetate. After hydrolysis of the dracetate, the A bond E may be introduced as described above, whereby there is formed 17:- ethinyl-I,4,6-a;ndrostafriene-l1B,17E-diol-3 r I one (VIH). H0 The lr-keto compound, l-7u-ethi nyl 1,4;6-andrdstatriene-l'lfi-ol gl intone may be prepared By any of the followingmetho'dsr v g M CHECK Q'l)"0xidation ofi' compound with eh'r'omic an- 1 2) 60 hydride in pyridinei 1 (2 Employing the sequence" or reactions from I to n material. In this case, in view of the stability or the 0H lilfl hydroxyl. group in" the presence of a 1 7'w-ethin'yl pare: the rotective acetatewf:the 17fi=hydroxylIcompound before? brominatinge The: 1 1"-ket6 compoundcfiresfiond ing to III, i. e. 17 a-ethinyl-4-andrdstene-1713-01-3,1 l-di'o'flb;

anhydride p-toluenpsl'ilfonic acid tion of the l7-keto group. Reaction of II with potassium using 4-and-rostene'-=3,1 :-1,l'i trione as the starting 3 may first be brominated and subsequently dehydrobrominated to form the corresponding 4,6-androstadiene. This, in turn, may finally be subjected to microbiological dehydrogenation with B. sphaericus, for example, so as to introduce the A bond yielding 17tx-ethinyl-l,4,6-androstatriene-17/3-ol-3,1 l-dione.

Compounds of the general formula containing a 90!.- fluoro group are prepared as follows: 7

N-brolnoacetamide OH I Meson Potassium Acetate Hydrogen,

Fluoride XIV 17oz ethinyl 1,4-androstadiene 115,175 diol 3-one (IV) is converted to 1,4,9(11)-androstatriene am by dehydration of .the-ll-hydroxyl with an agent such as methane sulfonyl chloride. IX is then hydroxy-brominated with N-bromoacetamide or N-bromosuccinimide to yield the 9d-brorno-1lfl-hydroxy-diene (X). Dehydrobromination with a base such as potassium acetate follows to form the 95,11fl-epoxy compound (XI). 7 Brominetion at 06 (compound XII) isetfected through the use of an agent such as N-bromosuccinimide, followed by dehydrobromination by refluxing in a tertiary base such as lutidine to obtain 17a-etl1inyl-9;8,l1B-epoxy-l,4,6-androstatriene-l7fl-ol (XIII). The addition of hydrogen fluoride; efiects transformation to gd-flllOI'O-170t-6thiDyl-1,4,6- androstatriene-l1,3,l7B-diol-3-one (XIV).. When XIV is oxidized in a conventional manner with chromic anhydride inacetic acid, the corresponding11-keto compound of this invention (XV) is obtained. y

The intermediate IX, is alternatively prepared by reacting v 17a-ethinyl- 4-androstene-11B,17 3-diol-3-one (III) with an agent such as methane sulfonyl chloride-to form 17a ethinyl-4,9(11'.)wandrostadiene-17/3-ol-3 one, which may then be subjected to the action of B. sphaericus to obtain 17a-ethinyl-1,4,9 1 l ')'-andros tatiiene-17B-ol-3-one x The foregoing is more fully described in the following examples which are not to be construed as limiting the scope of the invention, but are given for illustrative purposes only.

EXAMPLE 1 1 7-Ethinyl-1,4,6-A ndrostatriene-l 1 18,1 7B-diol-3-one A. 3-PYRROLIDINO-3,5-ANDRO STADIENE-llB-OL-lT-ONE B. I'I-ETHINYL-l-ANDROSTENE-11B,17B-DIOL-3-ONE A sample of 260 mg. of potassium is dissolved in 25 ml. of boiling redistilled t-amyl alcohol. This solution is added dropwise with stirring at room temperature to a solution of 150 ml. of anhydrous ether saturated with acetylene. After five minutes of stirring, a solution in 150 ml. anhydrous ether of 2 g. of the product of Example 1, Procedure A is added dropwise over 1 hour while acetylene is bubbled through the stirred mixture. Bubbling and stirring is continued for 2 hours more. Dilute acetic acid containing 0.5 g. of sodium acetate is added cautiously. The mixture is boiled 20 minutes and then extracted with ether. The ether solution is washed neutral with sodium bicarbonate solution and water, dried and evaporated. The residue is crystallized from acetone-methanol to give 17-ethinyl-4-androstene-115,175- di0l-3-one.

C. l't-ETHINYL-1,4-ANDROSTADIENE-11fi,17B-DIOI.-

Bacillus sphaericus var. fusiformis (A. T. C. C. 7055) is incubated on nutrient agar for 24 hours at 28 C. One loopful of the culture is then transferred to 100 ml. of sterile 1% yeast extract (Basamin-Busch) of pH 6.8. The inoculated culture is incubated on a shaking machine for 6 hours at 28 C. and the resultant broth culture is used as a standard inoculum at a level of 1 percent.

The standard inoculum is added to each of ten 300 ml. shake flasks containing respectively 100 ml. of 1% yeast extract at pH 6.8. Growth of the organism'is followed turbidimetrically and 17-ethinyl 4 androstene-11 3,17,8-

diol-3-0ne prepared as above (50 mg. in 1 ml. of methanol/flask) is added at the peak of the growth curve. The transformation proceeds rapidly and is followed adequately by paper chromatography or polarography of aliquots from the shake flasks. When the substrate is completely transformed (12-24 hours), the contents of the remaining flasks are pooled, extracted exhaustively with chloroform, and the extracts are concentrated to a residue. The residual material is recrystallized from acetone to give 17-ethinyl-1,4-androstadiene-1113,17/3-di0l-3- one.

D. 17-ETHINYL-1,4-ANDROSTADIENE-11B,17B-DIOL-3- ONE 11,17-DIACETATE To a solution of 5 g. of the product of above procedure C in 50 ml. of acetic anhydride and 50 ml. of glacial acetic acid is added 0.5 g. of p-toluenesulfonic acid. The reaction mixture is allowed to stand at room temperature overnight and is then treated with water. The resulting precipitate is removed by filtration, chromatographed on magnesiumsilicate eluting with 1:1 ether-hexane, and crystallized from ether-hexane yielding 17-ethinyl-1,4- androstadiene-l 1,6,17B-diol-3-one 11,17-diacetate.

E. 6-BROM0-17-ETHINYL-1,4-ANDROSTADIENE-I1BJ7B- DIOL-3-ONE 11,17-DIACETATE' A sample of 2 g. of the product obtained in the above procedure D is dissolved by boiling in m1. of chlorobenzene and 501311. of carbontetrachloride and the solutlon is dried by distilling off ml. of solvent- To the solution is added 1188 g. of Nbromosuccinimide and the mixture is irradiated with a ZOO-watt ph'otoflood lamp while refluxingfor 15 minutes while succinimide crystallizesout. The mixture is cooled and washed with water, and the organic solution is dried, filtered and evaporated in vacuo to a residue which is 6-bromo-17-ethinyl-1,4- androstadiene-llml'lfidiol-one 11,17-diacetate.

=1. 17-ETHINYIAI,4;6-A:NDROSTA'I'RIENE IIBJTB-DIOD3- ONE ILIT-DIACETATE To 15 ml. of refluxing dry 'y-collindine is added 0.5 g.

of the product of procedure B. After 30 minutes boiling during which solid-precipitates, the mixture is cooled, poured into ice and water and the pH adjusted to 4-6 with dilute hydrochloric acid. The mixture is extracted three times with 25 ml. of methylene chloride,. and the solution is washed with water, dried, filtered and evaporated to dryness. The residue, is dissolved in a minimum of methylene chloride and chromatographed on activated magnesium silicate using hexane to develop the column. The fraction. eluted with. ether is the 17-ethinyl-1,4,6-ai1drostatriene-l1p,l7fl-diol-3-one 11,17-diacetate, which may be crystallized further from acetone-hexane.

A mixture is prepared of 1 g. of yeast extract concentrate and 1 ml. each of 2 M. potassium dihydrogen phosphate and 2 M. disodium phosphate in each 100 ml. Ten Erlenmeyer flasks (300 ml.) containing 100 ml. each are sterilized and inoculated with Flavobacterium dehydrogenans var. hydrolyticum. The flasks are shaken at 30 C. for 16 hours, and to each is added a solution of 50 mg. of the 11, 17-diacetate of l7-ethinyl-l,4,6-androstatriene- 1 lfl,l7fl-diol 3-one in 5 ml. of methanol. The cultures are shaken'at 30 C. for 24 hours and the combined broths are extracted three times with 300 ml. of methylene chloride and the extract is dried, filtered and evaporated to dryness. The residue is crystallized from acetone-hexane to give 17-ethinyl-1,4,6-androstatriene-1118,17B-diol-3-one. Alternatively, the compoundof this example may be prepared using the same reactions outlined above, but in a different sequence: -In the alternate method, the compound of procedure B (l7-ethinyl-4-androstene-115,17/3- dio1 -3 -one) is acetylated with acetic anhydride to give: the

corresponding '11,1'7-diacetate. This, in turn, is reacted 1 with N-bromosuccinimide to obtain the 6-bromo 4-androstene. Dehydrobromination of this compound is efiected by refiuxin'g'with a tertiary base such as collidine to yield 17-ethinyl-4,6-androstadiene-11,8,17/8-diol-3-one 11,17-diacetate. This l1,17-diacetate is hydrolyzed to the corresponding ll,l7-diol when treated with Flavobacterium dehydrogens var. hydrolyticum. The resulting diol is then subjected to the action of Bacillus sphaericus var. fusiformis to obtain the desired compound 17-ethiny1- 1,4,6-androstatriene-1 1p, l7 8-diol-3-one.

EXAMPLE 2 1 7 -ethz'nyl-1 ,4,6-androstatriene-I 7[3-0l-3,1 I-dione Two grams of the product of Example 1 is dissolved in 20 ml. of pyridine and added with stirring to a cooled slurry of 1 g. of chromic anhydride in 20 ml. of pyridine. The mixture is stirred overnight at room temperature, and then diluted with 40 m1. of 10% aqueous sodium sulfite, followed by stirring for 2 hours. The mixture is acidified with aqueous sulfuric acid and extracted with methylene chloride. This solution is washed neutral with water, dried, filtered and evaporated to a residue. The product, 17- ethinyl-1,4,6-androstatriene-17p-ol-3,ll-diol, is obtained by crystallization of the residue from acetone-hexane.

Alternatively, the compound of this examplemaybe' prepared via the following series of reactions; A. 3-PYRRoLIDINO-a5-ANDROSTAJJIENE-auJ'I-TRIONE 4-and'rostene-3,l1,17-trione is reacted in the manner described in Example 1, procedure A to yield 3-pyrrolidino- 3,5-androstadiene-l1,17-dione.

B. 17 -ETHIN YL--ANDROSTENEa17B-OL-3JLDION E The pyrrolidino-androstadiene prepared above is re acted with acetylene under the conditions outlined in Example l, procedure B. Crystallization of the residue from acetone-methanol gives l7-ethinyl 4 androstene-17p-ol- 3,11-dione.

C. 17-ETHINYL-1,4-ANDROSTADIENE-l'lB-OLlJLDIONE The product of the above procedure B is subjectedto the action of Bacillus sphaericus var. fusiformis as described in Example 1, procedure C. The residual material is recrystallized from acetone to give l7-ethinyl-l,4-androstadiene- 1 7/3-ol-3,1 l-dione.

D. 6-BROM0-1'l-ETHINYL-L-ANDROSTADIENE-l'IB-OL- 3,11-DIONE 2 grams of 17-ethinyl-1,4-androstadi'ene-17fl-ol 3,1ldione is brominated with 1.15 grams of N-bromosuccinimide in the manner described in Example 1, procedure E. The resulting residue is 6-bromo-17-ethinyl-l,4-androstadiene-l7/3-ol-3,l1-dione with an ultraviolet spectrum having A max=245 m E. 17-ETHINYIr1,4,6-ANDROSTATRIENE-17B-0L-3,11-

' i I DIONE A sample of 2 grams of the 6-bromo-l,4-androstadiene compound prepared above is refluxed with 'y-collidine and purified in the manner described. in Example 1, procedure F. The fraction eluted with ether is the desired 17-ethinyl- 1,4,6-andr0statriene-17 3-01-3,1l-dione which may be crystallized further from acetone-hexane. The ultraviolet spectrum has max: 223, 255, 297 m The sequence of the reactions in the synthesis of the compound of this example is not critical. Thus, we may alternatively prepare this compound by reacting l7 ethinyl 4 androstene-N B-013,1l-dione (product or procedure B in Example 2) with N bromosuccinimide to obtain 6-bromo-17-ethinyl-4-androsteue-l7p+ol-3',1'1-dione. This, in turn, when refluxed with a-tertiary amine. such as. 'y-collidine yields 17-ethinyl-4,6-androstadiene-l'l'fi ole 3,1l-dione. The action. of Bacillus sphaericus var. fusi formis then converts this androstadiene to the desired compound, 17-ethinyl 1,4,6 --androstatriene 17fl ol-3,11 dione. I

EXAMPLE 3 A. 17-ETHINYL-1,4,9 (11)-ANDROSTATRIENE-l7B-Olr3- ONE A sample of 5 grams of l7-ethinyl-1,4-androstadiene- 11,8,17B-dio1-3-one (product of procedure C, Example 1) is dissolved in 20 m1. of dimethylformamide and 4 ml. of dry pyridine. The solution is chilled and stirred, and 4.1 g. of methane sulfonyl chloride in 41 ml. of dimethylformamide is added.

After being stirred at room temperature for 20 hours, the mixture is poured into 200 ml. of water, and extracted twice with ml. of methylene chloride. The organic layer is washed with 10% sodium bicarbonate and with water, dried and evaporated. The residue is crystallized from aqueous acetone to give 17-ethinyl-1,4,9(11)-androstatriene-l7 8-ol-3-one.

B. 9a-BROMO-17 -ETHINYL-l,4-ANDROSTADIENE-11B,17B- DIOL-3-ONE A sample of 2.5 g. of the product of above procedure A is dissolved in 250 ml. of dry tetrahydrofuran and 25 m1. of water. The solution is chilled with stirring to 5 C. and 2.5 g. of N-bromoacetamide is added. After the addition of 25 ml. of 1 N perchloric acid, the mixture is allowed to stand at room temperature for 48 hours.

The solution is diluted with 10% sodium sulfite and water and extracted with 200 ml. of chloroform twice. The extract is washed with water, dried and evaporated giving a residue which is dissolved in acetone, treated with charcoal, filtered and diluted with hexane. The product, 9a-bromo-l7-ethinyl-1,4-androstadiene-l13,17,3- diol-3-one, is recrystallized from acetonehexane.

C. 17-ETHINYL-9B,11B-OXIZDO-lA-ANDROSTADIENE- 17B-0Ir3-0NE A sample of 2 g. of the 9a-bromo product of above procedure B is dissolved in 200 ml. of acetone and 4 g. of potassium acetate is added. The solution is refluxed for 24 hours and evaporated to a residue in vacuo. Crystallization from acetone-hexane gives the desired epoxide, 17-ethinyl-9,8,1lfi-oxido-1,4-androstadiene-17/8- ol-3-one.

D. 6-BROMO-17-ETHINYL-9B,11B-0XIDO-1A ANDRO- STADIENE-17fi-0L-3-ONE A sample of 2 g. of the epoxide of above procedure C is dissolved by boiling in 100 ml. of chlorobenzene and 50 ml. of carbon tetrachloride, and the solution is dried by distilling off 5 ml. of solvent. To the solution is added 1.19 g. of N-bromosuccinimide and the mixture is irradiated with a 200-watt photoflood lamp and refluxed for 15 minutes while succinimide crystallizes out. The mixture is cooled, filtered, and the filtrate washed with water. The organic solution is dried over sodium sulfate, filtered, and evaporated in vacuo to a residue of 6-bromo- 17 ethinyl-9,6,1lp-oxido-l,4-androstadiene-17B-ol-3-one.

E. 17-ETHI-NYL-9B,11B-OXIDO-1,4,6-ANDROSTATRIENE- 17B-OL-3-ONE To 30 ml. of refluxing dry 2,4-lutidine is added .1.0 g. of 6-br0mo-17-ethinyl-9fl,11/8-oxido-l,4-androstadiene- 17p-ol-3-one. After 30 minutes boiling during which solid precipitates, the mixture is cooled, poured into ice and water, and the pH adjusted to 4-6 with dilute hydrochloric acid. The mixture is extracted three times with 25 ml. of methylene chloride, and the solution is washed with water, dried, filtered and evaporated to dryness.

The residue is dissolved in a minimum of methylene chloride and chromatographed on activated magnesium silicate using haxane to develop the column. The fraction eluted with ether is 17-ethinyl-9/8,115-oxido-1,4,6- androstatriene-17;3-ol-3-one which may be crystallized further from acetone-hexane.

F. 9a-FLUORO-17-ETHINYL-1,4,6-ANDROSTATRIENE- 11fi,17B-DIOL-3-ONE A sample of 1 g. of the product of above procedure E is dissolved in 100 ml. of anhydrous, alcohol-free chloro form and chilled to in a Dry Iceacetone bath. The stirred solution is treated with 1 g. of anhydrous hydrogen fluoride for 15 hrs. when the solution is washed with sodium bicarbonate solution, dried, filteredand evaporated. The residue is chromatographed on activated magnesium silicate and the fraction eluted with ether is crystallized from acetone-hexane .to give 9a-fll10I'0-17- ethinyl-1,4,6-androstatriene-1 15,17,3-diol-3-one.

In the same manner, use of hydrogen chloride and hydrogen bromide in place of hydrogen fluoride, leads to 9a-chloroand 9a-bromo-17-ethinyl-1,4,6-androstatriene-1lfl,l7fi-diol-3-one respectively.

EXAMPLE 4 9u-flu0r0-17-ethinyl 1,4,6 androstatriene-I7fl-ol-3,11-

dione A sample of 0.5 g. of 9a-fluoro-17-ethinyl-l,4,6-androstatriene-l15,17B-diol-3-one is dissolved in 15 ml. of acetic acid, stirred and treated with a solution of mg. of chromic anhydride in 0.5 cc. of water to which 1.0 cc. of acetic acid is added. The solution is stirred 15 minutes, treated with methanol, stirred 30 minutes and poured into water. The precipitate is collected, dried and recrystallized from acetone-hexane to give 9a-flu0r0-17-ethinyl- 1,4,6-androstatriene-17 3-01-3,1 l-dione.

We claim:

1. Unsaturated steroids having the following formula:

2. 17 ethinyl 1,4,6-androstatriene 1113,1713 diol 3 one.

3. 17-ethinyl-1,4,6-androstatriene-17fl-ol-3,1l-dione. 4. 9u-fluoro-l7-ethinyl 1,4,6 androstatriene-llfi,17fidiol-3-one.

5. 9u-fluQI0 17 ethinyl 1,4,6-androstatriene-17fl-ol- 3,1 l-dione.

No references cited- 

1. UNSATURATED STEROIDS HAVING THE FOLLOWING FORMULA: 